Composition for improving memory, learning ability, and cognitive ability

ABSTRACT

There is provided a composition for improving memory, learning ability, and cognitive ability. It has been confirmed that a peptide having a C-terminal region ended to GAG had an effect of improving the memory. In order for the peptide to have the effect, it has been confirmed that the peptide should be a peptide of which the length consists of at least 4 amino acids. Further, it has been confirmed that a peptide of which the length of the peptide having the C-terminal region ended to GAG consists of 5 to 9 amino acids has the same effect. As a result, the peptide of the present invention can be used as the composition for improving memory, learning ability, and cognitive ability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of the U.S. applicationSer. No. 15,482,752, filed Apr. 8, 2017, the disclosure of which isincorporated herein in its entirety by reference.

TECHNICAL FIELD

The present invention relates to a composition for improving memory,learning ability, and cognitive ability.

BACKGROUND

The brain has various functions, but the most important function ismemory and cognition. If there are no cognitive and memory abilities inthe human, it is difficult to perform everyday life and it becomes aproblem for survival. The memory and cognition are associated withalmost all functions of the brain and brain structures associated withthe memory and cognition are various and closely connected to almost allbrain parts.

The memory are classified into several steps and divided into a step ofregistering and encoding information, a storing step, and a step ofaccessing and withdrawing to a place of memory.

The encoding refers to an initial process in which information enteredinto the brain through sensory organs is learned and memorized. Theinformation is first stored through the encoding, but in order for thestored information to be continuously retained and stored more firmly, aprocess after encoding is required and this process is calledconsolidation. If the consolidation of the memory is not achieved well,memory forgetting occurs rapidly and memory retention becomes difficult.The withdrawal means a process of consciously invoking the contentsstored in the long-term memory. The withdrawal method includes recallingand recognition. The recalling is to invoke consciously the contents ofthe memory and the recognition is to invoke the contents while hints areapplied. In most cases, the recalling is more difficult than therecognition. However, like patients with frontal lobe injury orsubcortical vascular dementia, the recalling is difficult, but therecognition is achieved well, and in this case, the encoding and thestoring of the memory are performed well, but there is failure of thewithdrawal. If there is a memory storage failure, both the recalling andthe recognition have the failure.

In addition, short-term memory is also referred to as working memory,which is a process of performing the next task by using the informationafter storing the information for a short period. The short-term memorymeans a temporary stay before the information entering the brain hardensto the long-term memory. A feature of the working memory is workingmemory that is usually erased after performing a predetermined task.

The long-term memory means learning a new task and memorizing the newtask again after a predetermined time elapses. Memorizing things whichhave been experienced in our daily lives or the contents which have beenlearned again after the time elapses corresponds to almost the long-termmemory.

The memory and cognitive disorders correspond to very serious diseasesthat make daily life impossible, and includes diseases caused by a widevariety of causes and mechanisms, such as aging, Alzheimer's disease,schizophrenia, Parkinson's disease, Huntington's disease, pick disease,Creutzfeldt-Jakob disease, depression, aging, head injury, stroke, CNShypoxia, cerebral ischemia, encephalitis, forgetfulness, traumatic braininjury, hypoglycemia, Wernicke-Korsakoff syndrome, drug addiction,epilepsy, fasciola hepatica, hippocampal sclerosis, headache, brainaging, dementia, frontotemporal lobar degeneration, tumor, normalpressure hydrocephalus, HIV, cerebrovascular disease, cerebral disease,cardiovascular disease, amnesia, radiation exposure, metabolic disease,hypothyroidism, mild cognitive impairment, cognitive deficiency andattention deficit. In order to solve the memory and cognitive disorders,the related art has made various efforts, but up to now, there have beenno reports and application on the efficacy of synthetic peptides havingprecise sequences as well as substances having an excellentneuroprotective effect and a brain function improving effect againstvarious brain-nervous system diseases.

SUMMARY

The present invention has been made in an effort to provide a peptidefor improving memory, learning ability, and cognitive ability.

In order to achieve the above object, an exemplary embodiment of thepresent invention provides a peptide including an amino acid sequencewith a C-terminal region of GAG.

In an exemplary embodiment of the present invention, preferably, thepeptide is derived from a silk fibroin hydrolyzate, but is not limitedthereto.

In another exemplary embodiment of the present invention, preferably,the peptide is artificially synthesized, but may not be limited thereto.

In yet another exemplary embodiment of the present invention,preferably, the peptide has the number of amino acid residues of 4 to 6,but is not limited to the length, and the peptide has an amino acidsequence of GGAG, AGAG, QGAG, or SGAGAG, but may not be limited to theamino acid sequence.

In still another exemplary embodiment of the present invention,preferably, the peptide has the number of amino acid residues of 5 to 9,and the peptide has an amino acid sequence of QAGAG, SGGAG, orGAGGAGGAG, but may not be limited thereto.

The peptide of the present invention has excellent stability by itself,but in order to more largely improve the stability, various protectiongroups may be bound. Examples of the protection groups include an aminoacid group, an acetyl group, a fluorenyl methoxycarbonyl group, a formylgroup, a palmitoyl group, a myristyl group, a stearyl group andpolyethylene glycol (PEG). The protection groups may be bound to variousamino acid residues of the peptide of the present invention, butpreferably may be bound to N- or C-terminals.

Furthermore, the present invention provides a pharmaceutical compositionfor preventing or treating a memory, cognitive, or learning disorder,including the peptide of the present invention as an active ingredient.

In an exemplary embodiment of the present invention, preferably, thememory, cognitive, or learning disorder is a memory, cognitive, orlearning disorder caused by aging, Alzheimer's disease, schizophrenia,Parkinson's disease, Huntington's disease, pick disease,Creutzfeldt-Jakob disease, depression, aging, head injury, stroke, CNShypoxia, cerebral ischemia, encephalitis, forgetfulness, traumatic braininjury, hypoglycaemia, Wernicke-Korsakoff syndrome, drug addiction,epilepsy, fasciola hepatica, hippocampal sclerosis, headache, brainaging, dementia, frontotemporal lobar degeneration, tumor, normalpressure hydrocephalus, HIV, cerebrovascular disease, cerebral disease,cardiovascular disease, amnesia, radiation exposure, metabolic disease,hypothyroidism, mild cognitive impairment, cognitive deficiency andattention deficit, but may not be limited thereto.

The composition may include a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier included in the composition isgenerally used in formulation, and includes lactose, dextrose, sucrose,sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate,gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose,methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate,mineral oil, and the like, but may not be limited thereto. Thepharmaceutical composition may further include lubricants, wettingagents, sweeteners, flavors, emulsifiers, suspensions, preservatives,and the like in addition to the ingredients.

The pharmaceutical composition may be administered orally orparenterally. In the case of the parenteral administration, intravenousinjection, subcutaneous injection, muscle injection, intraperitonealinjection, endothelial administration, topical administration,intranasal administration, intrapulmonary administration, rectaladministration, and the like may be performed.

During oral administration, since a protein or a peptide are digested,the oral composition may be formulated by coating an active agent or tobe protected from decomposition in the stomach. Further, the compositionmay be administered by any apparatus in which an active substance maymove to a target cell.

A suitable dose of the pharmaceutical composition may be variouslyprescribed by factors such as a formulation method, an administrationtype, age, weight, and gender of a patient, a pathological condition,food, an administration time, an administration route, an excretionrate, and a response susceptibility. A preferable dose of thecomposition may be in a range of 0.001 to 100 mg/kg based on an adult.

The term “pharmacologically effective dose” means a dose suitable forpreventing or treating memory disorder, cognitive disorder or learningdisorder.

The composition is formulated by using a pharmacologically acceptablecarrier and/or excipient according to a method that may be easilyperformed by those skilled in the art to be prepared in a unit dosageform or prepared by intrusion into a multi-dose container. In this case,the formulation may be a form of solutions, suspensions, syrups oremulsions in oils or aqueous media or a form of extracts, discutient,powders, granules, tablets or capsules, and may additionally include adispersant or a stabilizer. Further, the composition may be administeredas an individual therapeutic agent or co-administered with othertherapeutic agents, and sequentially or simultaneously administered withtherapeutic agents in the related art.

Also, the present invention provides a food composition for enhancementof a brain or cognitive function, including the peptide of the presentinvention as an active ingredient.

In an exemplary embodiment of the present invention, preferably, thebrain or cognitive function is a learning ability, a memory ability, orconcentration, but may not be limited thereto.

An amount of the peptide in the food or drink of the present inventionmay be added with 0.01 to 15 wt % of the entire food weight, and ahealth drink composition may be added at a ratio of 0.02 to 5 g andpreferably 0.3 to 1 g based on 100 ml, but it may be easily determinedby those skilled in the art according to a product.

The food composition may further include a cytologically acceptable foodsupplementary additive in addition to the peptide and may be prepared ina form of tablets, capsules, pills, liquid preparations, jellies,powders, granules, and the like.

In the food composition of the present invention, other ingredients arenot particularly limited except for including the peptide as therequired ingredient, and like a general drink, various flavoring agents,natural starches, or the like may be included as an additionalingredient. Examples of the aforementioned natural carbohydrates includegeneral sugars, such as monosaccharides, for example, glucose, fructose,and the like; disaccharides, for example, maltose, sucrose, and thelike; and polysaccharides, for example, dextrin, cyclodextrin, and thelike, and sugar alcohols, such as xylitol, sorbitol, and erythritol. Asthe flavoring agent other than the above examples, natural flavoringagents (thaumatin, stevia extract (for example, rebaudioside A,glycyrrhizin, and the like) and synthetic flavoring agents (saccharin,aspartame, and the like) may be advantageously used. A ratio of thenatural carbohydrate may be generally about 1 to 20 g and preferablyabout 5 to 12 g per 100 ml of the composition of the present invention.

The food composition of the present invention may include variousnutrients, vitamins, minerals (electrolytes), flavoring agents such assynthetic flavoring agents and natural flavoring agents, coloring agentsand thickening agents (cheese, chocolate, and the like), pectic acid andsalt thereof, alginic acid and salt thereof, organic acid, a protectivecolloidal thickener, a pH adjusting agent, a stabilizer, a preservative,glycerin, alcohol, a carbonic acid agent used in a carbonated drink, orthe like, in addition to the ingredients. Besides, the food compositionof the present invention may include pulps for preparing natural fruitjuice and fruit juice drinks, and vegetable drinks. The ingredients maybe used independently or in combination. The ratio of the additives isgenerally selected in a range of 0 to 20 parts by weight per 100 partsby weight of the compound of the present invention.

Further, the present invention provides a polynucleotide encoding thepeptide of the present invention.

The “polynucleotide” is a polymer of a deoxyribonucleotide or aribonucleotide which is present in a form of a single strand or a doublestrand. The polynucleotide includes an RNA genome sequence, DNA (gDNAand cDNA) and an RNA sequence transcribed therefrom, and includesanalogs of natural polynucleotides unless specifically stated otherwise.

The polynucleotide includes not only the nucleotide sequence but also acomplementary sequence to the nucleotide sequence. The complementarysequence includes not only a completely complementary sequence but alsoa substantially complementary sequence. The sequence means a sequencewhich may hybridize with the nucleotide sequence under stringentconditions which are known in the art.

Further, the polynucleotide may be modified. The modification includesaddition, deletion, or non-conservative substitution or conservativesubstitution of the nucleotide. It is understood that the polynucleotideencoding the amino acid sequence includes a nucleotide sequence havingsubstantial identity with respect to the nucleotide sequence. Thesubstantial identity may be a sequence having homology of at least 80%,homology of at least 90%, or homology of at least 95%, in the case ofanalyzing a sequence which is aligned to maximally correspond to anydifferent sequence from the nucleotide sequence and aligned by using agenerally used algorithm in the art.

Furthermore, the present invention provides a recombinant vectorincluding the polynucleotide of the present invention.

The term “vector” means a means for expressing a target gene in a hostcell. For example, the vector includes a plasmid vector, a cosmidevector, and virus vectors such as a bacteriophage vector, an adenovirusvector, a retrovirus vector, and an adeno-associated virus vector. Thevector which may be used as the recombinant vector may be prepared bymanipulating plasmids (for example, pSC1O1, pGV1106, pACYC177, ColE1,pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14,pGEX series, pET series, pUC19, and the like), phages (for example,Agt4AB, A-Charon, A zl, M13, and the like) or virus (for example, CMV,SV40, and the like).

In the recombinant vector, the polynucleotide encoding the peptide maybe operatively linked with a promoter.

The term “operatively linked” means a functional binding between anucleotide expression regulatory sequence (for example, a promotersequence) and a different nucleotide sequence. Accordingly, theregulatory sequence may regulate transcription and/or translation of thedifferent nucleotide sequence by the functional binding.

The recombinant vector may be typically constructed as a vector forcloning or a vector for expression. The expression vector may usegeneral vectors which are used to express foreign proteins in plants,animals, or microorganisms in the art. The recombinant vector may beconstructed by various methods known in the art.

The recombinant vector may be constructed by using a prokaryotic cell oran eukaryotic cell as a host. For example, the used vector is anexpression vector, and in the case of using the prokaryotic cell as ahost, the vector generally includes a strong promoter (for example, apLA promoter, a trp promoter, a lac promoter, a tac promoter, a T7promoter, and the like), a ribosome binding site for initiation oftranslation, and a transcription/translation termination sequence. Inthe case of using the eukaryotic cell as a host, a replication originthat functions in the eukaryotic cell included in the vector includes anfl replication origin, an SV40 replication origin, a pMB1 replicationorigin, an adeno replication origin, an AAV replication origin, a CMVreplication origin, a BBV replication origin, and the like, but is notlimited thereto. Further, a promoter (for example, a metallothioneinpromoter) derived from a genome of a mammalian cell or a promoter (forexample, a adenovirus late-phase promoter, a vaccinia virus 7.SKpromoter, an SV40 promoter, a cytomegalovirus (CMV) promoter and a tkpromoter of HSV) derived from a mammalian virus may be used, and apolyadenylation sequence is generally used as a transcriptiontermination sequence.

Also, the present invention provides a host cell transformed with therecombinant vector of the present invention.

The host cell of the present invention may use any host cell known inthe art, and as the prokaryotic cell, for example, E. coli strains, suchas E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B,E. coli X 1776, and E. coli W3110, bacillus subtilis strains, such asbacillus subtilis and bacillus thuringiensis, and enterobacteriaceae andstrains, such as salmonella typhimurium, serratia marcesensis andvarious pseudomonas species are included. In the case of transformationto the eukaryotic cell, as the host cell, yeast (Saccharomycecerevisiae), insect cells, plant cells and animal cells, for example,SP2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK,COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, and the like may beused.

Further, the present invention provides a method for preparing thepeptide of the present invention including incubating the host cell ofthe present invention.

The insertion into the host cell of the polynucleotide or therecombinant vector including the polynucleotide may use an insertionmethod which is well-known in the art. The transfer method may use aCaCh method or an electroporation method, or the like when the host cellis the prokaryotic cell, and use microinjection, calcium phosphateprecipitation, electroporation, liposome-mediated transfection and genebombardment when the host cell is the eukaryotic cell, but is notlimited thereto.

A method of screening the transformed host cell may be easily performedby using a phenotype expressed by a selection marker according to amethod well-known in the art. For example, when the selection marker isa specific antibiotic resistance gene, a transformant may be easilyscreened by incubating the transformant in a medium including theantibiotic.

According to the present invention, it has been confirmed that a peptidehaving a C-terminal region ended to GAG had an effect of improving thememory. In order for the peptide to have the effect, it has beenconfirmed that the peptide should be a peptide of which the lengthconsists of at least 4 amino acids. Further, it has been confirmed thata peptide of which the length of the peptide having the C-terminalregion ended to GAG consists of 5 to 9 amino acids has the same effect.As a result, the peptide of the present invention can be used as acomposition for improving memory, learning ability, and cognitiveability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a memory enhancing effect of a peptideof which an amino acid sequence of a C-terminal region is GAG through apassive avoidance test. A y axis is time (sec).

FIG. 2 is a diagram illustrating a memory enhancing effect of a peptideof which an amino acid sequence of a C-terminal region is GAG through aY maze test. A y axis is spontaneous alternation (%).

FIG. 3 is a diagram illustrating a memory enhancing effect of a peptideof which an amino acid sequence of a C-terminal region consisting of 5to 9 amino acids is GAG through a passive avoidance test.

FIG. 4 is a diagram illustrating a memory enhancing effect of a peptideof which an amino acid sequence of a C-terminal region consisting of 5to 9 amino acids is GAG through a Y maze test.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detailthrough Examples. However, these Examples are to exemplify the presentinvention and the scope of the present invention is not limited to theseExamples.

Example 1: Synthesis of Peptides

Synthesized peptides were obtained from Genscript (New Jersey, USA). Thepeptides were synthesized by a flexpeptide technology method andconfirmed by using high pressure liquid chromatography and massspectrometry. Amino acid sequences of the synthesized peptides are aslisted in Table 1.

TABLE 1 Peptide name Amino acid (sequence number) sequence Peptide-I AGPeptide-2 GAG Peptide-3 AGAG Peptide-4 QGAG Peptide-5 GGAG Peptide-6SGAGAG

Further, the present inventors additionally synthesized peptides(sequence numbers 7 to 9) of amino acid sequences 5 to 9 to perform anadditional test. These synthesized peptides were obtained from Genscript(New Jersey, USA). The peptides were synthesized by a flexpeptidetechnology method and confirmed by using high pressure liquidchromatography and mass spectrometry. The amino acid sequences of thesynthesized peptides are as follows;

(sequence number 7) QAGAG (sequence number 8) SGGAG (sequence number 9)GAGGAGGAG

Example 2: Reagents and Animals

Scopolamine was purchased from Sigma-Aldrich (St. Louis, Mo., USA).4-week-old male ICR mice were purchased from Korean BioLink Co.(Chungbuk, Korea). After an adaptation period of one week, the mice wereused in a test and all reagents were administered intraperitoneally.Memory disorder was induced by injection of scopolamine before 30minutes of the test and the synthesized peptides were injected before 30minutes of the injection of scopolamine.

Example 3: Confirmation of Effect of Peptides in Passive Avoidance Test

A passive avoidance test was performed in the same bright room and darkroom. A floor of each room was formed in a shape in which 2-mm stainlesssteel rods were separated from each other at 1-cm intervals. Brightsquares (20×20×20 cm) had 100-W bulbs. These squares were connected to aguillotine door.

For an acquisition trial, the mouse was placed in the bright room afterinjection of the reagents and the door was opened after 10 seconds. Whenthe mouse completely entered the dark room, the door was closed andelectric shock was applied for 3 seconds. A retention trial wasperformed after 24 hours of the acquisition trial and the mouse waspositioned in the bright room. A latency time of the acquisition andretention trials was measured by a time until the mouse entered the darkroom of the box after the door was opened.

The retention latency in the passive avoidance test represents along-term memory function in rodents. Accordingly, an effect of a silkfibroin peptide on scopolamine-induced memory deterioration wasconfirmed by using a step-through passive avoidance test and the resultwas illustrated in FIG. 1.

As illustrated in FIG. 1, in the retention trial, a latency time of anormal saline-treated mouse was 180 seconds (maximum cut-off time). Itwas confirmed that an average of the step-through responses in ascopolamine-injected group with memory deterioration due to injection ofscopolamine was significantly lowered as compared with a normalsaline-treated group. Before administration of scopolamine, in groupsadministered with the synthesized peptides, in a peptide-I-administeredgroup consisting of two amino acids, slight improvement was achievedcompared with the scopolamine-administered group, and in apeptide-3-administered group, there was a little effect as compared withthe scopolamine-administered group. However, it was confirmed that inpeptide-2 and peptide-4 to 6-administered groups, the deterioratedmemory by scopolamine was improved to be close to a normal groupadministered with saline. From the above result, it was confirmed thatthere was a memory improving effect of the peptide of which theC-terminal region ended to GAG, and in order to have the effect, it isconfirmed that the peptide should be a peptide in which the lengthconsists of at least four amino acids.

As illustrated in FIG. 3, in the retention trial, a latency time of anormal saline-treated mouse was 180 seconds (maximum cut-off time). Itwas confirmed that an average of the step-through responses in ascopolamine-injected group with memory deterioration due to injection ofscopolamine was significantly lowered as compared with a normalsaline-treated group. Before administration of scopolamine, it wasconfirmed that in a group administered with the peptide consisting of 5to 9 amino acids of the present invention, the memory deteriorated byscopolamine was improved to be close to the normal group administeredwith saline. From the above result, the memory improving effect of thepeptide of which the C terminal region ended to GAG was confirmed.

Example 4: Confirmation of Effect of Peptide in Y Maze Test

The mouse was placed at one end of a Y maze with a length of one branchof 30 cm, a width of 5 cm, and a height of 13 cm and the order of entryinto each branch was recorded. Alternation was judged to be successfulif the mouse sequentially entered three different branches. Spontaneousalternation was defined as Equation below.

Spontaneous alternation (%)=the number of alternations/(total number ofentries−2)×100

As illustrated in FIG. 2, it was confirmed that the mean of spontaneousalternations of a scopolamine-injected group with memory deteriorationdue to the injection of scopolamine was significantly lower than that ofthe normal saline group. In groups administered with the synthesizedpeptides before administration of scopolamine, it was confirmed that ina peptide-I and 2 administered group consisting of 2 and 3 amino acids,slight improvement was achieved as compared with thescopolamine-administered group, but in a peptide-3 to 6 administeredgroup consisting of the number of amino acids of 4 to 6, the memorydeteriorated by scopolamine was significantly improved. From the aboveresult, it was confirmed that there was a memory improving effect of thepeptide of which the C-terminal region ended to GAG, and in order tohave the effect, it is confirmed that the peptide should be a peptide inwhich the length consists of at least four amino acids.

Further, as illustrated in FIG. 4, it was confirmed that the mean ofspontaneous alternations of a scopolamine-injected group with memorydeterioration due to the injection of scopolamine was significantlylowered as compared with the normal saline group. In the groupadministered with the peptide consisting of 5 to 9 amino acids of thepresent invention before administration of scopolamine, the memorydeteriorated by scopolamine was significantly improved, and in the groupadministered with peptides of sequence numbers 2 and 3, there was anexcellent effect as compared with a control group. From the aboveresult, the memory improving effect of the peptide of which the Cterminal region ended to GAG was confirmed.

What is claimed is:
 1. A polynucleotide encoding the peptide comprisingan amino acid sequence, wherein the amino acid sequence of the peptideis selected from the group consisting of SEQ ID NO:7(QAGAG), SEQ IDNO:8(SGGAG), and SEQ ID NO:9(GAGGAGGAG).
 2. The polynucleotide of claim1, wherein the polynucleotide is useful for preventing or treating amemory, cognitive, or learning disorder.
 3. The polynucleotide of claim1, wherein the memory, cognitive, or learning disorder is a memory,cognitive, or learning disorder caused by aging, Alzheimer's disease,schizophrenia, Parkinson's disease, Huntington's disease, pick disease,Creutzfeldt-Jakob disease, depression, aging, head injury, stroke, CNShypoxia, cerebral ischemia, encephalitis, forgetfulness, traumatic braininjury, hypoglycaemia, Wernicke-Korsakoff syndrome, drug addiction,epilepsy, fasciola hepatica, hippocampal sclerosis, headache, brainaging, dementia, frontotemporal lobar degeneration, tumor, normalpressure hydrocephalus, HIV, cerebrovascular disease, cerebral disease,cardiovascular disease, amnesia, radiation exposure, metabolic disease,hypothyroidism, mild cognitive impairment, cognitive deficiency orattention deficit.
 4. A recombinant vector comprising the polynucleotideof claim
 1. 5. A method of preparing a peptide comprising incubating ahost cell transformed with the recombinant vector of claim 4.